Biomedical Engineering Reference
In-Depth Information
toethanol or DTT to final concentration of 5% and 10 mM,re-
spectively). Centrifuge and apply the supernatant directly onto the
SDS-PAGE gel. The immunoprecipitated protein is identified by
staining, Western blot, and/or autoradiography.
References
Åkerström B, Björck L (1989) J Biol Chem 264:19740
Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring
Harbor Laboratory, p 447
4.10 Immunoelectrophoresis
Electrophoresis in agarose or agar gels is, in contrast to SDS-PAGE,
a nondenaturing method; therefore, the proteins move according to
their net charge, which is at the given alkaline pH mostly negative,
and are separated by electrophoretic mobility and not by molecular
size. The electropherograms obtained after immunoelectrophore-
sis are not comparable to SDS-PAGE pictures.
Solutions/Reagents
A
15.4 g sodium diethylbarbiturate (barbital sodium, barbitone
sodium, Veronal sodium), 2.76 g diethylbarbituric acid (bar-
bital), 1 g NaN
3
dissolved in ddH
2
O, pH adjusted to 8.4 and
filled up to 1000 ml
B
1.0 - 1.5% agar (w/v) in 1:1 diluted Soln. A. Melt before use in
a microwave oven or a boiling water bath
Pour 4 ml of molten Soln. B onto pre-coated slides (cf. Proto-
col 4.8.2). Punch wells according to Fig. 4.2 after gelation, and
remove residues of agar from the wells.
Mixtheprobe1:1withSoln.Aandaddatraceofbromophenol
blue. Fill the wells completely with sample.
Place the slide on the cooling plate of a horizontal electrophore-
sis apparatus, fill the tanks with 1:1 diluted Soln. B, and connect the
ends of the slide to the buffer tanks by wetted filter paper wicks. Run
the electrophoresis at 6 V
/
cmfor about 45 min.Stopelectrophoresis
when bromophenol blue is about 4 mm from the the wicks.
Cut the antiserum slots “Ab” after finishing the electrophoresis
and remove the agar within the slots. Pour antiserum into the
slot(s) and store the slide in a humid chamber at 37
◦
C for 4 - 6 h or
at 4
◦
C for 24 - 48 h. Visualize the precipitation lines as described
in Protocol 4.8.4.
References
Hudson L, Hay FC (1989) Practical immunology, 3rd ed. Blackwell, London,
p 236
Milford-Ward A (1977) In: Thompson RA (ed.) Techniques in clinical
immunology. Blackwell, Oxford, p 1
